The long-term objectives of this proposal are to define the role of phospholipases and heterotrimeric GTP-binding proteins in insulin secretion by the beta-cells of islets of Langerhans. The metabolism of glucose is sufficient for insulin secretion by islets of Langerhans. End-products of glucose metabolism in the beta-cell close K+ channels, which in turn depolarizes the beta-cell and causes influx of extracellular Ca2+ through voltage-dependent Ca2+ channels: this results in an increase in intracellular Ca2+ and subsequent insulin exocytosis. Simultaneously, stimulation of the beta-cell by fuel secretagogues or muscarinic agonists activates phospholipases C and A2, which are enzymes that hydrolyze phospholipids to generate lipid second messengers. Our ongoing studies have identified three isoforms of phospholipase A2 (type II secretory, cytosolic Ca2+-dependent, Ca2+-independent) in insulin- secreting beta-cells with different subcellular locations. In particular, insulin secretory granules are highly enriched in phospholipase A2. In addition, we have recently identified and localized the heterotrimeric G- binding protein Galphai to the insulin secretory granule by Western analysis and immuno-electron microscopy. Furthermore, stimulation of Galphai results in insulin secretion by permeabilized beta-cells. Preliminary experiments have identified three beta-cells proteins, p24, p50, and p95, as potential Galphai effector candidates. We suggest as a working hypothesis that activation of Galphai in the secretory granules is essential for insulin exocytosis. The questions to be addressed are: 1) what is the mechanistic role of Galphai in insulin exocytosis, and 2) is phospholipase A2 an effector of Galphai in insulin secretory granules? The specific aims of the proposal are as follows. AIM #1 is to elucidate the role of the secretory granule heterotrimeric G- protein, Galphai, in insulin exocytosis. In these experiments, a combination of biochemical, molecular, immunological, and cell biological approaches will be used to demonstrate that activation of Galphai is required for insulin secretion. AIM #2 is to identify the effectors coupled to the secretory granule heterotrimeric G-protein, Galphai. Two approaches will be used. First, we will determine whether phospholipase A2 is coupled to Galphai. In the second approach, we will determine the identity and role of the three proteins, p24, p50, and p95, that are coupled to Galphai following glucose stimulation of beta-cells.